The present invention concerns processes for the detection of the sensitivity of cells, e.g., tumor tissues, to the effects of human tumor necrosis factor (TNF) or lymphotoxin (LT). More particularly, the present invention concerns determining the sensitivity of cells, such as tumor cells, to the effects of TNF or LT by measuring the number of receptors on cells.
Hereinafter when TNF or LT are mentioned, applicants are describing molecules that are cytotoxic to cells and cause necrosis of tumors in mice.
Tumor necrosis factor (TNF) was observed by E. A. Carswell et al., Proc. Natl. Acad. Sci. USA, 72, 3666-3670 (1975) during a study of the antitumor activity of sera from mice infected with bacillus Calmette-Guerin (BCG) and subsequently injected with endotoxin. These sera have been observed to cause the hemorrhagic necrosis and regression of certain mouse tumors in vivo. These sera were also found to have cytotoxic/cytostatic effects on mouse and human tumor cells in vitro (E. A. Carswell et al, supra; L. Helson et al, Nature (London), 258, 731-732 (1975); D. N. Mannel et al, Infect. Immun., 28, 204-211 (1980); F. C. Kull and P. Cuatrecasas, J. Immunol., 126, 1279-1283 (1981); K. Haranaka and N. Satomi, Jpn. J. Exp. Med., 51, 191-194 (1981)). A similar factor was found to be induced in rats (Carswell et al, supra) and rabbits (Carswell et al, supra, N. Matthews, and J. F. Watkins, Br. J. Cancer, 38, 302-309 (1978)); J. Ostrove and G. E. Gifford, Proc. Soc. Exp. Biol. Medl, 160, 354-358 (1979).
The factor present in the sera of animals sensitized to BCG, or other immunopotentiating agents, such as Corynebacterium parvum or Zymosan, and then challenged with endotoxin that is capable of causing hemorrhagic necrosis of tumors in vivo has been termed tumor necrosis factor (TNF).
Biochemical characterization has shown that mouse serum TNF exists in at least two forms: a 150,000 M.sub.r form, (Kull and Cuatrecasas, supra and S. Green et al, Proc. Natl. Acad. Sci USA, 73, 381-385 (1976)) and a 40,000-60,000 M.sub.r form, D. N. Mannel et al, supra, Kull and Cuatrecasas, supra, and Haranaka, supra. TNF in rabbit serum has been reported to have a molecular weight of 39,000, N. Matthews et al, Br. J. Cancer, 42, 416-422 (1980) and 67,000 (M. R. Ruff and G. E. Gifford, J. Immunol., 125, 1671-1677 (1980)).
Studies have indicated that both in vivo and in vitro activities of mouse TNF appear to be a property of the same molecule. The cellular source of TNF in the mouse was initially assumed to be the macrophage, because the agents used to prime for TNF production cause massive hyperplasia of macrophages in liver and spleen, (Carswell et al, supra). From studies of macrophage-rich cell populations in vitro, N. Matthews, Br. J. Cancer, 38, 310-315 (1978) and D. N. Mannel et al, supra, a similar conclusion was reached with regard to the source of mouse and rabbit TNF. Direct evidence that macrophages are at least one cell type in the mouse capable of producing TNF comes from studies with cloned lines of mouse histiocytomas. These cells constitutively produce low levels of TNF that are greatly increased after exposure to endotoxin.
B. D Williamson et al, Proc. Natl. Acad. Sci. USA, 80, 5397-5401 (1983), described the capacity of human cell lines of hematopoietic origin to produce a factor with TNF activity. Evidence demonstrating that the molecule is a human TNF included the following: (1) the anticellular responses of a panel of human cell lines to human TNF, e.g., TNF(LuKII), or mouse TNF are indistinguishable and can be potentiated in a synergistic fashion by interferon, (2) mouse L cells made resistant to mouse TNF are resistant to human TNF, e.g., TNF(LuKII), (3) mouse L cells made resistant to human TNF, e.g., TNF(LuKII), are resistant to mouse TNF, and (4) human TNF, e.g., TNF(LukII) causes hemorrhagic necrosis of meth A sarcoma in the standard in vivo TNF assay.
Human tumor necrosis factor (TNF) produced by the LuKII cell line has been purified to a specific activity of 1.5.times.10.sup.7 units/mg of protein. Examination of this material by sodium dodecyl sulphate polyacrylamide gel electrophoresis demonstrates the presence of seven protein bands with molecular weights ranging from 80,000 daltons to 19,000 daltons. Peptide mapping analysis, as well as studies using monoclonal antibodies to human TNF, have demonstrated that all of the proteins present in these TNF preparations are related.
Heretofore there was no rapid in vitro method to predict whether a tumor will be capable of responding to TNF and LT.